基于PTEN/PI3K/AKT信号通路探讨补肾活血方改善慢性肾脏病矿物质和骨代谢异常大鼠的作用机制OA

Objective To observe the intervention effect of Bushen Huoxue Formula on rats with chronic kidney disease-mineral bone disorders(CKD-MBD),and to explore the mechanism by which Bushen Huoxue Formula improves CKD-MBD through the phosphatase and tensin homolog(PTEN)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.Methods Network pharmacology was used to predict the core targets and target enrichment pathways of Bushen Huoxue Formula in treating CKD-MBD,and the results of network pharmacology were verified by animal experiments.A total of 102 SPF-grade male SD rats aged 6-8 months were randomly divided into a normal control group,a model group,low-,medium-,and high-dose Bushen Huoxue Formula groups,and a Sevelamer group according to the random number table method,with 17 rats in each group.During weeks 1 to 4 of the experiment,rats in the model group,Bushen Huoxue Formula groups,and Sevelamer group were administered 250 mg/kg adenine suspension by gavage once daily,and were simultaneously fed a high-phosphorus diet.During weeks 5 to 8,adenine administration was switched to every other day by gavage.After successful modeling,rats in the low-,medium-,and high-dose Bushen Huoxue Formula groups received daily intragastric administration of Bushen Huoxue Formula(6.25,12.50,25.00 g·kg-1,respectively),rats in the Sevelamer carbonate group received intragastric administration of Sevelamer carbonate(240 mg·kg-1).The normal control group and model group received an equal volume of high-purity water by gavage,once daily.After 8 weeks of administration,serum renal function indices and calcium-phosphorus metabolism indicators were detected.Hematoxylin-eosin(HE)staining,Masson's trichrome staining,and periodic acid-Schiff(PAS)staining were used to observe the pathological changes in the glomeruli,renal tubules,and renal interstitium.HE staining and Alizarin red staining were used to observe the formation of calcified nodules in the rat thoracic aorta.HE staining was used to observe the bone tissue structure.Bone mineral density and cortical bone morphometry were measured by Micro-CT.Western blot and immunohistochemistry were used to detect the expression of PTEN/PI3K/AKT signaling pathway-related proteins in the aorta and bone tissue,respectively.Results The network pharmacology results showed that Bushen Huoxue Formula and CKD-MBD shared 276 common targets.KEGG enrichment analysis involved the PI3K/AKT and other signaling pathways.Compared with the normal control group,the rats in the model group showed declined renal function,disordered calcium-phosphorus metabolism,glomerular atrophy,renal tubular edema,and renal interstitial fibrosis in the renal tissue.Obvious calcified nodule formation was observed in the aorta;the bone tissue showed increased disconnection and separation of trabeculae,increased numbers of osteoblasts and osteoclasts on the trabecular surface,proliferation of fibrous tissue around the trabeculae,and reduced fat vacuoles;the trabecular mineral density(TMD)significantly reduced,and the pore volume fraction(po.V/TV)significantly increased.The protein expression of α-smooth muscle actin(α-SMA)was significantly downregulated,while the protein expression of bone morphogenetic protein-2(BMP-2)and runt-related transcription factor 2(Runx2)significantly increased in the model group(P<0.01).The protein expression of PTEN in the aortic and bone tissues significantly decreased,and the p-PI3K/PI3K and p-AKT/AKT ratios significantly increased(P<0.05).Compared with the model group,the Bushen Huoxue Formula medium-and high-dose groups showed improvements in renal function and calcium-phosphorus metabolism indicators(P<0.01,P<0.05),an increase in the number of normal glomeruli and renal tubules,reduced tubular inflammatory infiltration,alleviation of aortic calcified nodules,a decrease in po.V/TV(P<0.05),and decreased ratios of p-PI3K/PI3K and p-AKT/AKT in the aorta(P<0.05,P<0.01).The Bushen Huoxue Formula low-,medium-,and high-dose groups exhibited increased protein expression of α-SMA and decreased protein expression of BMP-2 and Runx2(P<0.05,P<0.01),with alleviated trabecular bone hyperplasia.Conclusion Bushen Huoxue Formula exerts a therapeutic effect on CKD-MBD rats,and the mechanism may be related to the up-regulation of PTEN expression in the PTEN/PI3K/AKT signaling pathway and the inhibition of PI3K/AKT signaling pathway phosphorylation.